1. Field of the Invention
The invention is in the field of antineoplastic agents and cancer diagnostic agents. More particularly, the invention pertains to novel 2-deoxyglucose conjugates, their use and methods of making such compounds.
2. Related Art
One of the biochemical “hallmarks” of malignancy is enhanced tumor glycolysis, which is primarily due to the overexpression of glucose transporters (GLUTs) and the increased activity of mitochondria-bound hexokinase in tumors (Medina, R. A. and Owen, G. I. (2002) Glucose transporters: expression, regulation and cancer. Biol. Res. 35, 9-26). Utilizing these cancer signatures, [18F] 2-fluoro-2-deoxyglucose (18FDG) based positron emission tomography (ET) has become a widely used molecular imaging modality in the detection of a wide range of human cancers (Czemin, J. and Phelps, M. E. (2002) Positron emission tomography scanning: current and future applications. Ann. Rev. Med. 53, 89-112).
18FDG is an analog of glucose that enters cells via glucose transporters (GLUT) and is phosphorylated to 18FDG-6-phosphate by hexokinase, the first enzyme in the glycolytic pathway. This enzyme converts glucose, 2-deoxyglucose or 18FDG from a neutral, membrane-permeable form to an anionic, membrane-impermeable form. Reversal of this reaction requires glucose-6-phosphatase, which is generally not present at high enough concentration in most cells to mediate this reaction. Further metabolism of 18FDG-6-phosphate by phosphoglucose isomerase, the next enzyme in the glycolytic pathway, or by enzymes in the glycogen or pentose shunt pathways does not occur (Pauwels, E. K., Ribeiro, M. J., Stoot, J. H., McCready, V. R., Bourguignon, M., and Maziere, B. (1998) FDG accumulation and tumor biology. Nuclear Med. Biol. 25, 317-322). Therefore, 18FDG-6-phosphate is trapped in the cell. The high affinity of 18FDG for tumors derives from the high levels of aerobic glycolysis (Vaupel, P., Kallinowski, F., and Okunieff, P. (1989) Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res. 49, 6449-6465) and overexpression of GLUTs exhibited by most, but not all (Weinhouse, S. (1976) The Warburg hypothesis fifty years later. Z. fur Krebsforsch. Klin. Onkol. 87, 115-126), tumors.
Speizer in 1985 (Speizer, L., Haugland, R., and Kutchai, H. (1985) Asymmetric transport of a fluorescent glucose analogue by human erythrocytes. Biochim. Biophys. Acta 815, 75-84), exploited the glucose transport pathway to deliver a fluorescent analog of glucose, 6-deoxy-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-aminoglucose, into human erythrocytes. This analog is a derivative of 6-deoxyglucose containing a fluorophore on the 6-position. A fluorescent derivative of 2-deoxyglucose, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) (Ex: 475 nm; Em: 550 nm), was introduced by Yoshioka et al. in 1996 (Yoshioka, K., Takahashi, H., Homma, T., Saito, M., Oh, K. B., Nemoto, Y., and Matsuoka, H. (1996) A novel fluorescent derivative of glucose applicable to the assessment of glucose uptake activity of Escherichia coli. Biochim. Biophys. Acta 1289, 5-9), who demonstrated that localization of this agent in the yeast Candida albicans was inhibited by D-glucose, but not L-glucose. Similar D-glucose (but not L-glucose) inhibited uptake of 2-NBDG has been demonstrated in E. coli (Yoshioka, K., Takahashi, H., Homma, T., Saito, M., Oh, K. B., Nemoto, Y., and Matsuoka, H. (1996) A novel fluorescent derivative of glucose applicable to the assessment of glucose uptake activity of Escherichia coli. Biochim. Biophys. Acta 1289, 5-9; Yoshioka, K., Saito, M., Oh, K. B., Nemoto, Y., Matsuoka, H., Natsume, M., and Abe, H. (1996) Intracellular fate of 2-NBDG, a fluorescent probe for glucose uptake activity, in Escherichia coli cells. Biosci. Biotech. Biochem. 60, 1899-1901; and Natarajan, A. and Srienc, F. (2000) Glucose uptake rates of single E. coli cells grown in glucose-lirnited chemostat cultures. J. Microbiol. Methods, 42, 87-96), yeast (Natarajan, A. and Srienc, F. (2000) Glucose uptake rates of single E. coli cells grown in glucose-limited chemostat cultures. J. Microbiol. Methods, 42, 87-96), vascular smooth muscle cells (Oh, K. B. and Matsuoka, H. (2002) Rapid viability assessment of yeast cells using vital staining with 2-NBDG, a fluorescent derivative of glucose. Intl. J. Food Microbiol. 76, 47-53), and pancreatic β-cells transfected to overexpress Glut2 glucose transporters (Lloyd, P. G., Hardin, C. D., and Sturek, M. (1999) Examining glucose transport in single vascular smooth muscle cells with a fluorescent glucose analog. Physiol. Res. 48, 401-410).
Utilization of the glucose transport system was further supported by competitive inhibition of 2-NBDG uptake by the glucose analogs 3-O-methyl glucose and D-glucosamine (Yoshioka, K., Saito, M., Oh, K. B., Nemoto, Y., Matsuoka, H., Natsume, M., and Abe, H. (1996) Intracellular fate of 2-NBDG, a fluorescent probe for glucose uptake activity, in Escherichia coli cells. Biosci. Biotech. Biochem. 60, 1899-1901). Internalization of 2-NBDG in isolated rabbit enterocytes was demonstrated by confocal microscopy (Roman, Y., Alfonso, A., Louzao, M. C., Vieytes, M. R., and Botana, L. M. (2001) Confocal microscopy study of the different patterns of 2-NBDG uptake in rabbit enterocytes in the apical and basal zone. Pflugers Arch.—Eur. J. Physiol. 443, 234-239). Conversion of 2-NBDG to 2-NBDG-6-phosphate in E. coli cells was confirmed by mass spectrometry and by demonstration that glucose-6-phosphatase regenerated 2-NBDG (Yoshioka, K., Saito, M., Oh, K. B., Nemoto, Y., Matsuoka, H., Natsume, M., and Abe, H. (1996) Intracellular fate of 2-NBDG, a fluorescent probe for glucose uptake activity, in Escherichia coli cells. Biosci. Biotech. Biochem. 60, 1899-1901). Localization of 2-NBDG in rat 9L glioma has recently been reported by Baidoo et al. (13aidoo, K. E., Mathews, W., and Wagner, H. N. (2000) Fluorescent imaging of deoxyglucose. 8th Intl. Conf: Peace through Mind/Brain Science Hamamatsu, Japan, February 2-4). These authors found that animals injected with 2-NBDG under fasting conditions (low serum glucose level) accumulated this probe in tumors, and uptake of 2-NBDG could be blocked under non-fasting conditions (high serum glucose level).
Additionally, U.S. Pat. No. 6,489,302 relates to a conjugate, comprising a saccharide and one or more therapeutic or diagnostic agents for treatment and diagnosis of cancer and viral disease which uses the GLUT pathway.
Near-infrared (NIR) dyes are presently attracting considerable interest as fluorescence probes for detection of cancer ((a) Lin, Y., Weissleder, R., and Tung, C. H. (2002) Novel near-infrared cyanine fluorochromes: synthesis, properties, and bioconjugation. Bioconjugate Chem. 13, 605-610. (b) Achilefu, S., Jimenez, H. N., Dorshow, R. B., Bugaj, J. E., Webb, E. G., Wilhelm, R. R., Rajagopalan, R., Johler, J., Erion, J. L. (2002) Synthesis, in vitro receptor binding, and in vivo evaluation of fluorescein and carbocyanine peptide-based optical contrast agents. J. Med. Chem. 45, 2003-2015. (c) Mujumdar, S. R., Mujumdar, R. B., Grant, C. M., and Waggoner, A. S. (1996) Cyanine-labeling reagents: sulfobenzindocyanine succinimidyl esters. Bioconjugate Chem. 7, 356-362) and as photosensitizers for cancer treatment by photodynamic therapy (PDT) (Dougherty, T. J., Gomer, C. J., Henderson, B. W., Jori, G., Kessel, D., Korbelik, M., Moan, J., and Peng, Q. (1998) Photodynamic therapy. J. Natl. Cancer Inst. 90, 889-905). Since tissue is relatively transparent to NIR light, NIR fluorescence imaging (NIRF) and PDT are capable of detecting and treating, respectively, even subsurface tumors. Owing to the need to increase photosensitizers' water solubility and to increase their affinity for tumor tissues, a great deal of effort has been devoted by many research groups to develop photosensitizers covalently linked with various carbohydrate moieties ((a) Sternberg, E. D., Dolphin, D. and Bruckner, C. (1998) Porphyrin-based photosensitizers for use in photodynamic therapy. Tetrahedron 54, 4151-4202. (b) Licha, K. (2002) Contrast agents for optical imaging. Top. Cur. Chem. 222, 1-29). However, none of them was intended to take advantage of the intracellular trapping mechanism for 2DG in tumor cells as it does for PDG.
A key limitation of cancer drugs is their lack of specificity for tumor cells. There is therefore a need to develop tumor-specific agents that are targeted at GLUT transporters, which constitute the main pathway that tumor cells utilize for import of substrates for energy production. Since glucose is the principal substrate for energy production utilized by most if not all tumors the agents which target this critical pathway should exhibit a substantially enhanced level of tumor specificity.
Stable bacteriochlorophyll (BChl) analogs derived from R. Sphaeroides are excellent NIR dyes for NIRF and PDT because of their favorable photophysical properties (1O2 yield: 45%) and long activation and fluorescence emission wavelengths (750-850 nm). (Pandey, R. K.; Zheng, G., Porphyrins as Photosensitizers in Photodynamic Therapy. In The Porphyrin Handbook, ed.; Kadish, K. M.; Smith, K. M.; Guilard, R., Eds. Academic Press: Boston, 2000; Vol. 6, pp 157-230.) Since native BChl is very unstable and undergoes rapid oxidation to the chlorin state (660 nm), preparation of a stable BChl analog is a synthetic challenge. In recent years, many approaches to remove the major points of fragility in the BChl a molecule have been tried by various investigators. These include replacing the central metal, magnesium, with other metal ions (e.g., palladium) to form stable complexes, (Fiedor, J.; Fiedor, L.; Kammhuber, N.; Scherz, A.; Scheer, H., Photodynamics of the bacteriochlorophyll-carotenoid system. 2. Influence of central metal, solvent and beta-carotene on photobleaching of bacteriochlorophyll derivatives. Photochemistry & Photobiology. 2002 August;76(2):145-52; Schreiber, S.; Gross, S.; Brandis, A.; Harmelin, A.; Rosenbach-Belkin, V.; Scherz, A.; Salomon, Y., Local photodynamic therapy (PDT) of rat C6 glioma xenografts with Pd-bacteriopheophorbide leads to decreased metastases and increase of animal cure compared with surgery. International Journal of Cancer. 2002 May 10;99(2):279-85.) modifying the isocyclic ring, and replacing the phytyl group at the propionyl residue either through transesterification or by conversion to the corresponding amide derivatives. (Rosenbach-Belldn, V.; Chen, L.; Fiedor, L.; Tregub, I.; Paviotsky, P.; Brumfeld, V.; Salomon, Y.; Scherz, A., Serine conjugates of chlorophyll and bacteriochlorophyll: photocytotoxicity in vitro and tissue distribution in mice bearing melanoma tumors. Photochemistry & Photobiology. 1996 July;64(1):174-81.) In particular, Pandey, et al. have shown that naturally occurring unstable BChl a (extracted from Rb. Sphaeroides) can be converted to the stable bacteriochlorins, bacteriopurpurin-18 and bacteriopurpurinimide. (Kozyrev, A. N.; Zheng, G.; Zhu, C. F.; Dougherty, T. J.; Smith, K. M.; Pandey, R. K., Syntheses of stable bacteriochlorophyll-a derivatives as potential photosensitizers for photodynamic therapy. Tetrahedron Letters 1996, 37, (36), 6431-6434; Chen, Y.; Graham, A.; Potter, W.; Morgan, J.; Vaughan, L.; Bellnier, D. A.; Henderson, B. W.; Oseroff, A.; Dougherty, T. J.; Pandey, R. K., Bacteriopurpurinimides: highly stable and potent photosensitizers for photodynamic therapy. Journal of Medicinal Chemistry. 2002 Jan. 17;45 (2):255-8.) In this approach, converting the fused isocyclic ring to a cyclic imide moiety enhanced the stability and solubility of the BChl analogs. Attachment of a variable allyl substituent to the imide moiety allowed the lipophilicity of the molecule to be fine-tuned. Some of these agents are highly potent against radiation-induced fibrosarcoma (RIF-1) in mice. In another elegant demonstration, Fiedor et al. found that inserting palladium into the BChl ring significantly improves its stability toward reactive oxygen species. (Fiedor, J.; Fiedor, L.; Kammhuber, N.; Scherz, A.; Scheer, H., Photodynamics of the bacteriochlorophyll-carotenoid system. 2. Influence of central metal, solvent and beta-carotene on photobleaching of bacteriochlorophyll derivatives. Photochemistry & Photobiology. 2002 August;76(2):145-52.) One such compound, Tookad®, is now under clinical evaluation for treating prostate cancer. To achieve higher levels of tumor selectivity, target-specific BChls derived from stable BChl suitable by bioconjugation are desired. For this purpose, a universal linker introduced into a stable BChl molecule that will facilitate binding of biomolecules such as peptides, proteins, and other affinity ligands is desirable. Accordingly, the present invention provides synthesis of novel functionalized BChl dyes containing an isothiocyanate moiety, their bioconjugatioh to cancer targeting agents, and the ini vivo optical imaging of animal tumors with these new bioconjugates.